1. Field of the Invention Generally, the invention relates to the field of analyte separation and protein detection, referred to commonly as proteomics. More particularly, the invention relates to the separation of analytes and detection of separated proteins using microfluidics and chip-based technologies.
2. Description of the Related Art
A key step to proteomic research is the ability to separate, isolate and independently detect proteins, i.e., hormones, enzymes, antibodies, etc. or more basically, peptides, polypeptides, amino acids and the like, within an analyte or sample (hereafter “analyte”).
Heretofore, the most-widely used, but often most technically challenging, proteomic separation method is 2-dimensional isoelectric focusing (“IEF”)—polyacrylamide gel electrophoresis (“PAGE”) or IEF-PAGE. IEF separates proteins according to their net charge i.e., isoelectric point (“pI”) in wide pH range ampholyte buffers. The polyacrylamide gel acts as a sieve and separates analytes by Stokes' radii, i.e., molecular weight. The gel is stained to identify high and moderate abundance proteins forming spots. These spots, especially spots that change position relative to control gels, can be cut out, the proteins extracted and characterized. Extraction efficiency is on the order of 7%. Given this low extraction efficiency, IEF-PAGE is an unreliable process for protein separation.
A second approach to protein separation is high performance liquid chromatography (“HPLC”). During HPLC, mobile phase buffers under high pressure, force an approximately 1-10 μl volume of analyte through the narrow confines of specially coated, immobile silica particles. Separations are based on hydrophobicity e.g., “reversed phase” using, for example, octadecal alkane [C-18] or other hydrophobic materials linked to silica with gradients of mobile phases such as water-acetonitrile, charge i.e., relative binding to strong cationic or anionic charged groups attached to silica, and size i.e., size exclusion chromatography. HPLC is expensive, slow and inefficient, requiring relatively large sample and solvent amounts.
Yet a third separation technique, capillary electrophoresis (“CE”), separates nanoliter (“nl”) amounts of analytes according to their mass/charge (“m/z”) ratios. A strong electrical potential difference pulls the analyte through an ampholyte zwitterion buffer in a long (e.g., 30 cm) capillary tube. While this technique is excellent for separation of less than 100 peptides, the ampholytes can interfere with detection processes such as ultra-violet (“UV”) absorbance and mass spectrometry-mass spectrometry (“MS-MS”) analysis.
Further, several miniaturized ampholyte-free modifications of CE have been developed including capillary isoelectric focusing (“CIEF”) where the analytes, but not the buffer, are mobilized and separated according to pI and base stacking within the capillary tube, where NaOH or another suitable base is loaded after the sample. The electrical potential difference pulls the NaOH and any charged proteins towards the opposite end of the capillary tube. A pH gradient forms as the migrating NaOH is diluted. Individual proteins become immobilized in a narrow band at the point where they have a net neutral charge. UV-Visible (“UV-Vis”) and photodiode array (“PDA”) absorbance detectors can continuously monitor the separation and optimize molecular resolution, i.e., separation, to 0.002 pI units. Capillary tubes can be packed with gels or other column support materials, but these can be difficult to manufacture in a reproducible manner.
In addition to CIEF, other separation techniques which are variations on CE include separation based on size and charge differences between analytes which is termed Capillary Zone Electrophoresis (“CZE”) or Free Solution CE (“FSCE”), separation of neutral compounds using surfactant which form into micelles which is called micellar electrokinetic capillary chromatography (“MECC”) or sometimes referred to as (“MEKC”) and sieving of solutes through a gel network commonly referred to as Capillary Gel Electrophoresis (“GCE”). Capillary electrochromatography (CEC) is an associated electrokinetic separation technique that involves applying voltages across capillaries filled with silica gel stationary phases. Separation selectivity in CEC is a combination of both electrophoretic and chromatographic processes.
Microfluidic separations are largely based on first, the electrophoretic mass transfer of the analyte and second, separation of the proteins within the analyte. The gel and ampholytes of IEF-PAGE described above for electrophoretic mass transfer are eliminated in the microfluidic microchannel and microcapillary methods. Further, reversed phase and other organic separation methods are of limited applicability when combined with a CE-based second separation step unless the solvent(s) are miscible in the CE buffer (e.g. 15% methanol buffers) and further do not separate as distinct bands, do not interfere with analyte separations or wall interactions, have low background for the detectors and are amenable to electrospray MS-MS. Separations that work at large dimensions often do not work in Microsystems.
There is a need in the art for a system and method for high throughput protein separations on a micro-scale that facilitates two-dimensional separation that produces protein components that are easily detectable by recognized detector configurations.